Human gastric cancer antigen gene and gastric cancer antigen protein

ABSTRACT

A tumor antigen gene is identified by screening a cDNA library derived from a gastric cancer cell line that can induce gastric cancer antigen specific cytotoxic T cell (CTL) by means of hybridization and PCR utilizing an amino acid sequence of peptide fragment of a known gastric cancer antigen protein, introducing a selected cDNA clone into a cell of gastric cancer cell line that cannot induce gastric cancer antigen specific CTL so that the clone should be expressed in the cell, and selecting a transgenic cell that has acquired the ability to induce CTL. According to the present invention, there are provided a protein capable of inducing immune response against human gastric cancer, DNA encoding the protein, as well as vaccine for treatment and prevention of human gastric cancer, and agent for treatment and prevention of human gastric cancer.

TECHNICAL FIELD

The present invention relates to a protein capable of inducing acytotoxic T cell (Cytotoxic T Lymphocytes, see “Ika Men'ekigaku (MedicalImmunology), Revised 3rd Edition, Ed. by K. Kikuchi, also abbreviated as“CTL” hereinafter) against human gastric cancer cells in vivo or invitro, and a DNA encoding the protein. Particularly, the presentinvention relates to a protein capable of presenting CTL against humangastric cancer cells by being bound to HLA-A31 antigen (Human LeucocyteAntigen, see “Gendai Men'ekigaku (Current Immunology)”, 2nd Edition, Ed.By Y. Yamamura and T. Tada), and a DNA encoding the protein.

The present invention also relates to an agent for prevention ortreatment of human gastric cancer, which comprises a protein capable ofinducing CTL against human gastric cancer cells in vivo or in vitro, anda vaccine for prevention or treatment of human gastric cancer, whichcomprises a recombinant virus or a recombinant bacterium containing aDNA encoding the protein.

BACKGROUND ART

As the therapies for malignancy, in addition to surgical treatment,radiotherapy, and chemotherapy, there has been attempted immunotherapywhich aims at obtaining therapeutic effect by enhancing the immunefunction of host patients. However, most of the immunotherapy procedurespractically used thus far have obtained the effect by non-specificallyenhancing immunecompetence of host patients, and drugs capable ofinducing complete cure of tumors in clinical cases have not beenpractically used yet.

Many researchers have conducted investigations utilizing animal tumormodels mainly based on mice to develop a drug capable of completelycuring tumors. As a result, it has been clarified that tumors may becompletely cured by efficiently inducing antigen specific immuneresponses, in particular, inducing cytotoxic T cells (CTLs), againsttumor-associated antigens or tumor specific antigens expressed onvarious tumor cells. In order to treat tumors by such CTL induction, itis essential to elucidate an amino acid sequence of protein recognizedby tumor specific CTL and a DNA sequence encoding the protein for eachtumor.

In recent years, also for clinical tumors, it has been attempted toidentify an antigen inducing tumor specific CTL and a DNA encoding theantigen, and utilize them for therapy.

Based on such conception, tumor antigen proteins capable of inducing CTLand DNAs encoding such proteins have been searched for. However, thoseidentified so far are limited to the proteins such as those of MAGEfamily (T. Boon et al., Immunology Today, 18, 267-278, 1997), Mart-1,Thyrosinase, gp100 (S. A. Rosenberg, Immunology Today, 18, 175-182),which are the tumor antigens present in melanoma, and DNAs encodingthem, and the proteins present in epidermoid cancer (derived from humanhead and neck cancer, CASP-8, J. Exp. Med., 186, 785-793, 1997) and DNAsencoding them.

On the other hand, as for digestive tract cancers including gastriccancer, the presence of a tumor antigen peptide which can induce CTL hasbeen identified by the present inventors (Japanese Patent UnexaminedPublication [KOKAI] No. Hei 9-151200/1997). However, it has not beenclarified at all what kind of protein from which tumor peptide isderived, and much less structure of DNA encoding the tumor antigenprotein.

SUMMARY OF THE INVENTION

Accordingly, the object of the present invention is to provide 1) a DNAsequence encoding a protein which can induce immune response againsthuman gastric cancer, 2) a protein sequence encoded by theaforementioned DNA, 3) a vaccine for treatment or prevention of humangastric cancer which comprises a recombinant virus or a recombinantbacterium containing the aforementioned DNA, and 4) an agent fortreatment or prevention of human gastric cancer which comprises theaforementioned protein.

The present inventors noted that CTL recognizing tumor antigens playedan important role as a biological defense mechanism against tumor cells.That is, the present inventors noted that efficient induction of CTL byutilizing a protein usable as a part of vaccine or a DNA encoding such aprotein was effective for treatment and prevention of tumors, andconducted studies.

It has been known that the CTL induction against tumor antigens isachieved through specific expression of tumor antigen genes in tumorcells and presentation of antigenic peptides derived from the tumorantigen proteins on cell surfaces together with HLA antigens on tumorcells. Therefore, the present inventors attemped to identify a gene thatis specifically expressed in a gastric cancer cell line capable ofinducing CTL specific for gastric cancer cells.

As described above, the present inventors identified a tumor antigenpeptide (also referred to as “F4.2 peptide” hereinafter) which caninduce CTL against digestive tract cancers including gastric cancer, andelucidated its amino acid sequence (Japanese Patent UnexaminedPublication [KOKAI] No. Hei 9-151200/1997). Therefore, they attempted toisolate a cDNA encoding a protein from which the peptide is derived froma cDNA library of gastric cancer cell line by hybridization techniqueutilizing that amino acid sequence. As a result, several hybridizationpositive cDNA clones were identified. From these clones, those that canbe amplified by PCR utilizing primers corresponding to the sequence ofF4.2 peptide were further selected. When nucleotide sequences of allprovided clones were determined, however, any clone encoding a sequencecontaining an amino acid sequence exactly the same as F4.2 peptide wasnot found, while several clones containing partial sequences of F4.2peptide were selected.

On the other hand, other than the hybridization technique mentionedabove, the present inventors developed a method utilizing phenotypicalteration caused by gene transfer as an index as a method foridentifying a tumor antigen gene capable of inducing CTL specific for agastric cancer cell. That is, the present inventors conceived that atarget tumor antigen gene could be identified by introducing achromosome DNA fragment derived from a suitable cell line, or cDNAderived from gastric cancer cell line that can induce gastric cancerantigen specific CTL, into a cell line that cannot induce CTL so thatthe gene should be expressed in the cell, and selecting a transgenicclone that has acquired the ability to induce CTL.

The present inventors has already successfully established a CTL cellline (Tc-HST-2) derived from a gastric cancer patient, whichspecifically respond to tumor antigen restricted to HLA-A31, and agastric cancer cell line (HST-2) derived from the same patient, which isrecognized by the CTL cell line (J. Immunol. Meth., 154; 235-243, 1992,Cancer, 75; 1484-1489, 1995). The gastric cancer cell line expressesHLA-A31 antigen. They have also succeeded in establishing a cell line(HOBC8-A31 cell) that expresses HLA-A31 antigen, but is not recognizedby Tc-HST2 cells, and can be cultured in vitro. It is not expected thata gastric cancer antigen gene that can be expressed by HST-2 isexpressed in that HOBC8-A31 cell.

When the aforementioned method utilizing phenotypic alteration as anindex was applied to a cDNA clone encoding an amino acid sequence whichcontained a part of F4.2 peptide by using the aforementioned cell lines,it was found that a protein encoded by the cDNA activated the CTL cellline Tc-HST-2. Further, it was found that the cDNA was very useful as anagent for treatment and prevention of gastric cancer.

The present invention has been completed based on these findings.

That is, the present invention provides:

(1) A DNA which encodes a gastric cancer antigen protein present in ahuman gastric cancer cell (also referred to as the “DNA of the presentinvention” hereinafter).

(2) The DNA of above (1), which encodes a gastric cancer antigen proteinwhich comprises an amino acid sequence of SEQ ID NO: 2.

(3) The DNA of above (1), which encodes a gastric cancer antigen proteinwhich comprises an amino acid sequence of SEQ ID NO: 2 havingsubstitution, deletion, insertion, addition or inversion of one orseveral amino acids, and which can activate a cytotoxic T cellrecognizing a gastric cancer antigen protein.

(4) The DNA of above (2) which comprises at least nucleotidescorresponding to the nucleotide numbers 46-534 of the nucleotidesequence of SEQ ID NO: 1.

(5) The DNA of above (2) which is hybridizable with a nucleotidesequence comprising at least nucleotides corresponding to the nucleotidenumbers 46-534 of the nucleotide sequence of SEQ ID NO: 1 under astringent condition, and which encodes a protein that can activate acytotoxic T cell recognizing a gastric cancer antigen protein.

(6) The DNA of above (5), wherein the stringent condition is a conditionin which washing is performed at 60° C., and at a salt concentrationcorresponding to 1×SSC and 0.1% SDS.

(7) The DNA of above (1), wherein the gastric cancer antigen proteinencoded by the DNA comprises a partially modified amino acid sequencefor realizing more efficient induction of a cytotoxic T cell recognizingthe protein.

(8) A DNA which encodes a peptide containing an amino acid sequencecomprising at least amino acids corresponding to amino acid numbers62-70 of SEQ ID NO: 2, and being able to activate a cytotoxic T cellrecognizing a gastric cancer antigen protein.

(9) A gastric cancer antigen protein which comprises an amino acidsequence of SEQ ID NO: 2.

(10) A gastric cancer antigen protein which comprises an amino acidsequence of SEQ ID NO: 2 having substitution, deletion, insertion,addition or inversion of one or several amino acids, and which canactivate a cytotoxic T cell recognizing a gastric cancer antigenprotein.

(11) A peptide which contains an amino acid sequence comprising at leastamino acids corresponding to amino acid numbers 62-70 of SEQ ID NO: 2,and which can activate a cytotoxic T cell recognizing a gastric cancerantigen protein.

(12) A vaccine for prevention or treatment of human gastric cancer,which comprises a recombinant virus or a recombinant bacteriumcontaining a DNA of any one of claims 1-8.

(13) An agent for prevention or treatment of human gastric cancer, whichcomprises the protein of above (9) or (10), or the peptide of above(11).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph representing activity of HOBC8-A31 cells introducedwith 0131cDNAc-98 for inducing CTL as to TC-HST-2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail hereinafter.

The DNA of the present invention is a DNA encoding a gastric cancerantigen protein present in a human gastric cancer cell. The DNA encodesa protein that is expressed in a human gastric cancer cell, and caninduce cytotoxic T cells targeting the gastric cancer cell whenexpressed in a cell expressing the same type HLA as that of the gastriccancer cell.

The method for obtaining the DNA of the present invention will beexplained hereinafter. <1> Identification of cancer antigen gene thatcan induce gastric cancer cell specific CTL

The DNA of the present invention can be identified by utilizing (1) ahuman gastric cancer cell line which can be cultured in vitro and ofwhich HLA type has already been identified, (2) a CTL cell line whichrecognize the gastric cancer antigen which may be expressed by theforegoing gastric cancer cell line in the context of T cell receptor-and HLA-antigen-restriction, and can be cultured in a large scale invitro, and (3) a cell line which expresses the same HLA as that of theaforementioned gastric cancer cell line, but does not express thegastric cancer gene, and can be cultured in a large scale in vitro. Thatis, the DNA of the present invention can be obtained by selection of thecDNA derived from the cell line (1) which is able to induce the CTLactivity of the CTL cell line (2) when the cDNA is introduced into thecell line (3).

As the human gastric cancer cell line which can be cultured in vitro andof which HLA type has already been identified, for example, the HST-2cell established by the present inventors can be used (J. Immunol.Meth., vol. 154, p 235-243 (1992); Jpn. J. Cancer Res., vol. 84, p906-913 (1993)). This cell line is a gastric signet ring cell lineestablished from cancerous ascites of a gastric cancer patient.

As the CTL cell line which exhibits cytotoxicity against theaforementioned cell line in the context of T cell receptor and HLAagntigen restriction, and can be cultured in a large scale in vitro, theTc-HST-2 cell also established by present inventors can be used (J.Immunol. Meth., vol. 154, p 235-243 (1992); Jpn. J. Cancer Res., vol.84, p 906-913 (1993)).

It is described in Cancer, vol.75, p1484-1489 (1995) that theaforementioned CTL cell specifically reacts with the HST-2 cell and thatthis CTL cell recognizes the HST-2 antigen (HST-2 cell specific antigen)in the context of HLA-A31 restriction.

As the cell line which expresses the same HLA antigen as that of theHST-2 gastric cancer cell line, more specifically the HLA-A31 antigen,which is utilized when the Tc-HST-2 cell recognizes the HST-2 cell, butdoes not express the gastric cancer gene, i.e., is not recognized by theTc-HST2 cell, and can be cultured in vitro, the HOBC8-A31 cellestablished by the present inventors can be used.

In order to obtain a cDNA library comprising the gastric cancer antigengene that can induce CTL activity of Tc-HST-2 cell, the HST-2 cell canbe used. That is, the HST-2 cell can be cultured under a usual culturecondition, for example, in RPMI1640 medium supplemented with fetalbovine serum, and washed with phosphate buffered saline (PBS), and mRNAcan be purified from the cultured cells. Then, a cDNA library can beconstructed from the mRNA by using a suitable vector. Techniques formRNA purification, construction of cDNA library, gene transfer and thelike are well known to those skilled in the art, and described inliterature well known to those skilled in the art, for example,Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning, ColdSpring Harbor Laboratory Press, 2nd edition and the like.

A cDNA clone of the gastric cancer antigen gene that can induce the CTLactivity of the Tc-HST-2 cells can be identified by introducing thecDNAs of the aforementioned cDNA library into HOBC8-A31 cells, andexamining if the transformed HOBC8-A31 cells can induce the CTL activityof Tc-HST-2 cells as a result of the gene transfer (CTL induction test).

If a transgenic HOBC8-A31 cell can induce the CTL activity or not can bedetermined by the chromic acid release test (Science, vol. 249, p283-287 (1990)) or the TNF release test (Immunogenetics, vol. 35, p145(1992)). In the TNF release test, transgenic cells are cultured at 37°C. for 48 hours, added with Tc-HST-2 cells, and further cultured for 12hours, and TNF activity released in the culture supernatant is measuredto detect the CTL induction activity.

The TNF activity can be measured by the method described inImmunogenetics, vol. 35, p145 (1992). That is, a TNF susceptive cell,for example, Wehi164 cells are mixed with a sample to be assayed(coculture of transformant cells and Tc-HST-2), cultured for 24 hours,then added with MTT (3-(4,5-dimethylthiazol-z-yl)2,5-diphenyltetrazolium bromide) solution, and further cultured for 3hours. Then, formazan produced by conversion from MTT in mitochondria ofthe Wehi164 cells is dissolved with acid propanol (e.g., propanolcontaining 0.01% hydrochloric acid), and its produced amount isdetermined by measuring absorbance (OD₅₇₀) to assay the TNF activity.

The method for identifying a cancer antigen gene which can induce theaforementioned gastric cancer cell specific CTL will be described indetail in the working examples hereinafter.

A DNA sequence of cDNA encoding a gastric cancer antigen gene capable ofinducing the CTL activity of the Tc-HST-2 cell, which is identified asdescribed above, can be determined in a conventional manner by using aDNA sequencer.

Specific examples of the DNA of the present invention include a DNAencoding a gastric cancer antigen protein having the amino acid sequenceof SEQ ID NO: 2 depicted in SEQUENCE LISTING. More specific examplesthereof include a DNA containing a nucleotide sequence comprising atleast nucleotides corresponding to nucleotide numbers 46-534 of SEQ IDNO: 1 depicted in SEQUENCE LISTING. This DNA was identified by theaforementioned method as will be described in the examples hereinafter,and it is well understood by those skilled in the art that any codonencoding each amino acid residue in the amino acid sequence encoded bythe DNA can be replaced with another equivalent codon.

The DNA of the present invention also include a DNA encoding a peptidehaving an amino acid sequence comprising at least amino acid residuescorresponding to the amino acid numbers 62-70 of the amino acid sequenceof SEQ ID NO: 2 depicted in SEQUENCE LISTING, more specifically, a DNAcontaining a sequence comprising, for example, nucleotides correspondingto the nucleotide numbers 229-255 of SEQ ID NO: 1. Such peptides alsocan induce the gastric cancer cell specific CTL like the protein of thepresent invention.

An Escherichia coli strain harboring a plasmid 0131cDNAc-98 containing aDNA having the nucleotide sequence shown in SEQ ID NO: 1 was given aprivate number AJ13489, and it was deposited at the National Instituteof Bioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (zip code305-8566, 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan) on Jul. 10,1998, as an accession number of FERM P-16895, and transferred from theoriginal deposit to international deposit based on Budapest Treaty onJun. 14, 1999, and has been deposited as deposit number of FERM BP-6759

Because the nucleotide sequence of the DNA having the nucleotidesequence shown in SEQ ID NO: 1 was elucidated by the present invention,such a DNA can be obtained by amplifying it from human chromosome DNA ora cDNA library derived from a gastric cancer cell line that can inducegastric cancer antigen specific CTL by PCR utilizing oligonucleotidesproduced based on that nucleotide sequence as primers, or by aconventional solid phase DNA synthesis technique. While the primers usedfor PCR are not particularly limited, oligonucleotides corresponding toregions upstream from the nucleotide number 46 and downstream from thenucleotide number 534 in the nucleotide sequence shown in SEQ ID NO: 1are preferred.

The DNA of the present invention may be a gene derived from achromosome, and may be interrupted with one or more introns so long asit encodes a gastric cancer antigen protein.

The DNA of the present invention may encode a gastric cancer antigenprotein comprising substitution, deletion, insertion, addition orinversion of one or several amino acid residues at one or more sites solong as the activities of the encoded a gastric cancer antigen protein,i.e., the properties that it is recognized by cytotoxic T cells specificfor the protein, and it can activate such cytotoxic T cells, are notdeteriorated. The number meant by the term “several” herein used mayvary depending on the positions in the higher order structures of theprotein, kinds and the like of amino acid residues. This is arisen fromthe fact that there are highly analogous amino acids such as isoleucineand valine, and substitutions among such amino acids do not greatlyaffect the higher order structures of proteins.

A DNA encoding a protein substantially the same as the aforementionedgastric cancer antigen protein can be obtained by modifying thenucleotide sequence by, for example, site-specific mutagenesis so thatthe encoded amino acid sequence should have substitution, deletion,insertion, addition or inversion of amino acid residues at certainpositions. Such a modified DNA as mentioned above can also be obtainedby other conventional mutagenesis treatments. Examples of suchtreatments include, for example, a method involving in vitro treatmentof DNA encoding a gastric cancer antigen protein with hydroxylamine orthe like, method involving a treatment of a microorganism, e.g., abacterium belonging to the genus Escherichia, harboring a DNA encoding agastric cancer antigen protein with ultraviolet irradiation or an agentusually used for mutagenesis treatments such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.

A DNA encoding a protein substantially the same as the gastric cancerantigen protein can be obtained by expressing such a mutated DNA insuitable cells, and examining if it encodes a protein having theactivities of the gastric cancer antigen protein using theaforementioned CTL induction test. A DNA encoding a proteinsubstantially the same as the gastric cancer antigen protein can also beobtained by isolating a DNA that is hybridizable with a DNA having anuclectide sequence comprising the nucleotides corresponding to thenucleotide numbers 46-534 of the nucleotide sequence of SEQ ID NO: 1depicted in SEQUENCE LISTING or a part thereof under a stringentcondition, and encodes a protein having activities of the gastric cancerantigen protein. The “stringent condition” herein used means a conditionwhere a so-called specific hybrid is formed, but any non-specific hybridis not formed. While it is difficult to numerically define thecondition, exemplary conditions include one allowing hybridization ofDNAs having a homology of 90% or more, but not allowing hybridization ofDNAs having a homology less than 90%, and conditions under which DNA'sare hybridized with each other at a salt concentration corresponding toan ordinary condition of washing in Southern hybridization, i.e., 60°C., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS.

While genes hybridizing under these conditions may contain those havingbeen introduced with a stop codon, those having lost their activitiesand the like, they can be readily eliminated by the CTL induction testmentioned above.

Such modifications of the gastric cancer antigen protein as mentionedabove may be one simply not impairing the activities of the gastriccancer antigen protein, but it is preferably one enabling more efficientinduction of cytotoxic T cells recognizing the protein.

A gastric cancer antigen protein can be produced by ligating the DNA ofthe present invention to a suitable expression vector, transforming acell such as BCG bacteria and bacteria belonging to the genusEscherichia with the vector, and culturing the resulting transformedcell. When a DNA having the nucleotide sequence shown in SEQ ID NO: 1 isused as the DNA of the present invention, a gastric cancer antigenprotein having the amino acid sequence shown in SEQ ID NO: 2 can beobtained. When a modified DNA according to the present invention such asthose mentioned above is used, a modified gastric cancer antigen proteincan be obtained.

Because the DNA of the present invention, for example, a DNA having theDNA sequence of SEQ ID NO: 1 depicted in SEQUENCE LISTING or a partthereof, or a protein or peptide encoded by the DNA can induce gastric.cancer cell specific CTL, it is extremely hopeful as a therapeutic orprophylactic agent for human gastric cancer. For example, bacteria suchas BCG bacteria that are transformed with a recombinant DNA obtained byintroducing the DNA of the present invention into a suitable vector, andviruses such as vaccinia virus that carry the DNA of the presentinvention integrated into their genomes are effectively used as a livevaccine for treatment and prevention of human gastric cancer.

Dosage and administration may be similar to those used for conventionalsmallpox vaccination, BCG vaccine and the like.

When the protein or peptide of the present invention is used as atherapeutic or prophylactic agent for human gastric cancer, it can beadministered 1) as it is, 2) together with a pharmaceutically acceptablecarrier and/or diluent, or 3) by injection or transdermal absorptionthrough spraying together with auxiliaries such as those mentionedbelow, if necessary. The carrier herein used include, for example, humanserum albumin, and the diluent includes PBS, distilled water and thelike.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further explained in more detail withreference to the following examples, but the present invention is notlimited to these examples.

EXAMPLE 1 Construction of cDNA Library From HST-2

HST-2 cells were cultured at 37° C. in RPMI1640 medium supplemented withfetal bovine serum. After 1×10⁸ HST-2 cells were washed with phosphatebuffered saline (PBS), the total RNA (1.9 mg) was extracted from thecells by using RNAgents Total RNA Isolation System (Promega company),and the mRNA (4 mg) was purified by using PolyATract system 1000(Promega) The purification was performed by according to the protocol ofPromega. cDNA (4 mg) was synthesized by using a cDNA synthesis kit(Amersham Pharmacia Biotech). The synthesized cDNA was ligated to BstX-1adapter, and integrated into an expression vector, PcDNAI/Amp (AmershamPharmacia Biotech). A cDNA library was constructed according to theprotocol of Pmersham Pharmacia Biotech.

EXAMPLE 2 Screening for cDNA Encoding Peptide F4.2 by Hybridization

Cloning of cDNA by hybridization was performed by using GENE TRAPPERcDNA Positive Selection System (GIBCO). The procedure therefor will beoutlined below. A degenerated primer encoding F4.2 (amino acid sequenceis shown in SEQ ID NO: 3), F4.2×(5′-TAC TCY TGG ATG GAY AT-3′: SEQ IDNO: 4), was synthesized and biotiylated. The cDNA library was digestedat sense sequence side with the digestive enzymes contained in theaforementioned kit, GENE II protein and Exonucease III, and thus madeinto single-stranded cDNA. The single-stranded cDNA was hybridized withthe aforementioned biotiylated primer, and added with magnetic beadscoated with streptavidin so that the cDNA hybridized to the biotiylatedprimer should be bound to the magnet beads through binding of biotin andstreptavidin. Single-stranded cDNA was collected from the magnet beads,and restored into double-stranded cDNA using the above primer which wasnot biotiylated. This double-stranded cDNA was inserted into a vectorpRC/RSV, and Escherichia coli was transformed with the obtainedrecombinant vector. In this manner, 3000 kinds of cDNA clones wereobtained.

EXAMPLE 3 Screening for cDNA Clone Havina F4.2 Sequence by PCR

All of the cDNA clones provided in Example 2 were screened for a clonewhich contained a DNA sequence encoding F4.2 by PCR utilizing primersshown below.

As the forward primer for the PCR, various kinds of oligonucleotideshaving various kinds of sequences including a nucleotide sequencecorresponding to the amino acid sequence of F4.2 (5′-TAC TCC TGG ATG GACATC-3′, SEQ ID NO: 5). As the reverse primer, an oligonucleotide whichhad a sequence corresponding to a region located about 50 bp downstreamfrom the insert of the vector (5′-TCT AGA TGC ATG CTC GAG-3′, SEQ ID NO:6) was used. The PCR was performed under the following conditions usinga PCR apparatus (Thermal cycler, Applied Biosystem).

First step: 95° C. for 5 minutes

Second step: 95° C. for 30 seconds

Third step: 55° C. for 30 seconds

Fourth step: 72° C. for 30 seconds

Fifth step: 72° C. for 7 minutes

Among the above-mentioned steps, the second step to the fourth step wererepeated for 30 times.

EXAMPLE 4 DNA Sequence Determination of cDNA Clone

Nucleotide sequences of the PCR products obtained in Example 3 weredetermined by using ABI PRISM 310 Genetic Analyzer (Applied Biosystem).The nucleotide sequence determination was performed according to theprotocol of Applied Biosystem. No clone containing a sequencecorresponding to the entire sequence of F4.2 was found among the cloneswhose sequences were determined. However, a clone having a sequencecorresponding to a partial sequence of F4.2 was found among the cloneswhose sequences were determined (0131cDNAc-98). This close was obtainedby using an oligonucleotide corresponding to the amino acid sequenceshown in SEQ ID NO: 5 as the forward primer. The nucleotide sequence ofthis clone containing a sequence corresponding to a partial sequence ofF4.2 is shown in SEQ ID NO: 1. The amino acid sequence that can beencoded by this sequence is shown in SEQ ID NO: 2.

EXAMPLE 5

Transformation of HOBC8-A31 Cell With 0131cDNAc-98 cDNA and Assay ofTransformant Cell for Ability to Induce Activation of Tc-HST-2CTL Cell

The cDNA obtained in Example 4 (0131cDNAc-98) was introduced into a cellline (HOBC8-A31) that did not express a gastric cancer gene to formtransiently transformed cells. A gastric cancer patient-derived CTL cellline that specifically respond to a gastric cancer antigen in thecontext of HLA-A31 restriction was sensitized by the above transformantcell, and TNF produced by the responded CTL cell was added to a highlyTNF-sensitive cell line. The ability of the transformant to induceactivation of CTL cell was assayed by using the cytotoxic activity ofTNF as an index.

The cDNA obtained in Example 4 (0131cDNAc-98) was introduced intoHOBC8-A31 cells by using ripofectin. The transgenic cells were culturedat 37° C. in RPMI1640 medium supplemented with 0.1 ml of 10% FCS. After48 hours, when transient gene expression was observed, 10³ of TcHST-2cells (gastric cancer patient-derived CTL cell line that specificallyresponded to a gastric cancer antigen in the context of HLA-A31restriction) were added to the cells, and co-cultured for 24 hours.

The cytotoxicity was assayed by the following MTT assay. That is, 7×10⁴cells of highly TNF-sensitive cell line Wehil64 (obtained from LudwigInstitute for Cancer Research, Brussels, Belgium) were suspended in a120 μl volume, introduced into wells of a microtiter plate, each addedwith 30 μl of the culture supernatant from the aforementionedco-culture, and cultured for 24 hours. Then, 5 mg/ml solution of MTT(3-(4,5-dimethylthiazol-z-yl) 2,5-diphenyltetrazolium bromide) was addedto each well, and then incubated for further three hours. Subsequently,150 μl of propanol containing 0.01% hydrochloric acid was added to eachwell to solubilize formazon produced during the cultivation, and thenTNF activity was determined by measuring absorbance at a wavelength of570 nm.

The results are shown in FIG. 1. HST-2 cells were used as a positivecontrol, and HOBCu-A31 cells transformed with a vector (PcDNAI) wereused as a negative control. As seen from the results shown in FIG. 1,CTL induction activity for TC-HST-2 was confirmed in the HOBC8-A31 cellsthat were introduced with 0131cDNAc-98 (FIG. 1).

When a sequence of the nucleotide numbers 229-255 of the nucleotidesequence of SEQ ID NO: 1 depicted in SEQUENCE LISTING was introducedinto cells and expressed in the same manner as described above, it wasconfirmed that the transformant cell line had the CTL induction activityfor TC-HST-2.

6 1 1093 DNA Homo sapiens CDS (46)..(534) 1 cagtgtgctg ggaaaggttcgaacacggca cccgcactgc gcgtc atg gtg cag gcc 57 Met Val Gln Ala 1 tgg tatatg gga cga cgc ccc ggg cga ccc gcg gca acc cca ccg ccc 105 Trp Tyr MetGly Arg Arg Pro Gly Arg Pro Ala Ala Thr Pro Pro Pro 5 10 15 20 cga ccccgg ccc gcc cag tgg ggc ctg gag cag ctg cgg cgg ctc ggg 153 Arg Pro ArgPro Ala Gln Trp Gly Leu Glu Gln Leu Arg Arg Leu Gly 25 30 35 gtg ctc tactgg aag ctg gat gct gac aaa tat gag aat gat cca gaa 201 Val Leu Tyr TrpLys Leu Asp Ala Asp Lys Tyr Glu Asn Asp Pro Glu 40 45 50 tta gaa aag atccga aga gag agg aac tac tcc tgg atg gac atc ata 249 Leu Glu Lys Ile ArgArg Glu Arg Asn Tyr Ser Trp Met Asp Ile Ile 55 60 65 acc ata tgc aaa gataaa ctt cca aat tat gaa gaa aag att aag atg 297 Thr Ile Cys Lys Asp LysLeu Pro Asn Tyr Glu Glu Lys Ile Lys Met 70 75 80 ttc tac gag gag cat ttgcac ttg gac gat gag atc cgc tac atc ctg 345 Phe Tyr Glu Glu His Leu HisLeu Asp Asp Glu Ile Arg Tyr Ile Leu 85 90 95 100 gat ggc agt ggg tac ttcgac gtg agg gac aag gag gac cag tgg atc 393 Asp Gly Ser Gly Tyr Phe AspVal Arg Asp Lys Glu Asp Gln Trp Ile 105 110 115 cgg atc ttc atg gag aaggga gac atg gtg acg ctc ccc gcg ggg atc 441 Arg Ile Phe Met Glu Lys GlyAsp Met Val Thr Leu Pro Ala Gly Ile 120 125 130 tat cac cgc ttc acg gtggac gag aag aac tac acg aag gcc atg cgg 489 Tyr His Arg Phe Thr Val AspGlu Lys Asn Tyr Thr Lys Ala Met Arg 135 140 145 ctt gtt tgt ggg aga accggt gtg gac agc gta caa ccg gcc cgc 534 Leu Val Cys Gly Arg Thr Gly ValAsp Ser Val Gln Pro Ala Arg 150 155 160 tgaccatttt gaagcccgcg ggcagtaccgtgaaattctg gcacagaccg cctagcatgc 594 tgcctgggaa ctaacacgcg cctcgtaaaggtcccaatgt aatgacttga gcagaaaatc 654 aatactttct ctttgctttt agaggatagctaggttatct ttcctttgta agattatttg 714 atcagaatat tttgtaatga aaggatctagaaagcaactt ggaagtgtaa agagtcacct 774 tcattttctg taactcaatc aagactggtgggtccatggc cctgtgttag ttcatgcatt 834 cagttgagtc ccaaatgaaa gtttcatctcccgaaatgca gttccttaga tgcccatctg 894 gacgtgatgc cgcgcctgcc atgtaagaaggtgcaatcct agataacaca gctagccaga 954 tagaagacac ttttttctcc aaaatgatgccttggggtgg ggagtggtag ggggaagagc 1014 tcccacccta aggggcacac actgagttgcttatgccact tcttgttcaa aataaagtaa 1074 ctgccttaat cttactttc 1093 2 163PRT Homo sapiens 2 Met Val Gln Ala Trp Tyr Met Gly Arg Arg Pro Gly ArgPro Ala Ala 1 5 10 15 Thr Pro Pro Pro Arg Pro Arg Pro Ala Gln Trp GlyLeu Glu Gln Leu 20 25 30 Arg Arg Leu Gly Val Leu Tyr Trp Lys Leu Asp AlaAsp Lys Tyr Glu 35 40 45 Asn Asp Pro Glu Leu Glu Lys Ile Arg Arg Glu ArgAsn Tyr Ser Trp 50 55 60 Met Asp Ile Ile Thr Ile Cys Lys Asp Lys Leu ProAsn Tyr Glu Glu 65 70 75 80 Lys Ile Lys Met Phe Tyr Glu Glu His Leu HisLeu Asp Asp Glu Ile 85 90 95 Arg Tyr Ile Leu Asp Gly Ser Gly Tyr Phe AspVal Arg Asp Lys Glu 100 105 110 Asp Gln Trp Ile Arg Ile Phe Met Glu LysGly Asp Met Val Thr Leu 115 120 125 Pro Ala Gly Ile Tyr His Arg Phe ThrVal Asp Glu Lys Asn Tyr Thr 130 135 140 Lys Ala Met Arg Leu Val Cys GlyArg Thr Gly Val Asp Ser Val Gln 145 150 155 160 Pro Ala Arg 3 10 PRTHomo sapiens 3 Tyr Ser Trp Met Asp Ile Ser Cys Trp Ile 1 5 10 4 17 DNAArtificial Sequence Description of Artificial Sequenceprimer 4tactcytgga tggayat 17 5 18 DNA Artificial Sequence Description ofArtificial Sequenceprimer 5 tactcctgga tggacatc 18 6 18 DNA ArtificialSequence Description of Artificial Sequenceprimer 6 tctagatgca tgctcgag18

What is claimed is:
 1. A DNA which encodes a gastric cancer proteinpresent in a human gastric signet ring cancer cell, wherein said proteincomprises an amino acid sequence of SEQ ID NO:2.
 2. The DNA according toclaim 1, which comprises at least nucleotides corresponding to thenucleotide numbers 46-534 of the nucleotide sequence of SEQ ID NO:
 1. 3.The DNA according to claim 1, which is hybridizable with a nucleotidesequence comprising at least nucleotides corresponding to the nucleotidenumbers 46-534 of the nucleotide sequence of SEQ ID NO: 1 under astringent condition, and which encodes a protein that can activate acytotoxic T cell recognizing a gastric cancer antigen protein.
 4. TheDNA according to claim 3, wherein the stringent condition is a conditionin which washing is performed at 60° C., and at a salt concentrationcorresponding to 1×SSC and 0.1% SDS.
 5. A DNA which encodes a peptideconsisting of amino acids 62-70 of SEQ ID NO:2, and being able toactivate a cytotoxic T cell recognizing a gastric cancer antigenprotein.
 6. A vector comprising the DNA according to claim
 1. 7. Avector comprising the DNA according to claim
 2. 8. A vector comprisingthe DNA according to claim
 3. 9. A vector comprising the DNA accordingto claim
 4. 10. A vector comprising the DNA according to claim
 5. 11. Ahost cell comprising the DNA according to claim
 1. 12. A host cellcomprising the DNA according to claim
 2. 13. A host cell comprising theDNA according to claim
 3. 14. A host cell comprising the DNA accordingto claim
 4. 15. A host cell comprising the DNA according to claim 5.